The human genome is under constant attack by endogenous and environmental DNA damaging agents. To assess the biological significance of exposure to environmental DNA damaging agents, it is necessary to understand the details of the complex response to DNA damage. Much less is known about the impact of environmental DNA damaging agents on the mitochondrial genome compared with the nuclear genome. In vertebrates, nuclear and mitochondrial forms of DNA ligase III? (LigIII?) are generated by alternative translation initiation. Most published studies have focused on the nuclear LigIII?/XRCC1 complex and the role of XRCC1 as a scaffolding factor that co-ordinates the activities of multiple repair factors. However, while the end processing factors PNKP, Tdp1 and aprataxin are all present in mitochondria, XRCC1 is not. We have found that LigIII? and Tdp1 form a stable complex when co-expressed in insect cells. In Aim 1, we will delineate the mechanisms underlying the participation of LigIII? in mitochondrial DNA metabolism. It is our working hypothesis that LigIII? and Tdp1 form the core complex that co-ordinates the repair of DNA single- strand breaks in mitochondria. Preliminary studies with the LigI/III inhibitor L67 have shown that cells with mitochondria are more sensitive to the ligase inhibitor and that this sensitivity is due to inhibition of mitochondrial (mito) LigIII?. Further studies revealed that inhibition of mito LigIII? has markedly different effects in cancer and non-malignant cells. In cancer cells, the ligase inhibitor caused increased mitochondrial superoxide (mito SOX) levels, reduced oxygen consumption, inhibition of autophagy and caspase 1-dependent apoptosis. By contrast, in non-malignant cells, the ligase inhibitor did not cause an increase in mito SOX or reduced oxygen consumption. However, oxidative phosphorylation was uncoupled and the cells became senescent. We hypothesize that non-malignant cells respond to L67-induced mitochondrial dysfunction by uncoupling oxidative phosphorylation and activating autophagy, thereby attenuating production of reactive oxygen species (ROS). In contrast, we hypothesize that cancer cells are unable to reduce mitochondrial ROS generation, resulting in the initiation of a vicious cycle in which rising ROS levels cause increased mitochondrial dysfunction that activates apoptosis. In Aim 2, we will elucidate the mechanisms underlying the differential effect of inhibiting mito LigIII? on mitochondrial DNA metabolism and oxidative phosphorylation in non-malignant and cancer cells. In Aim 3, we will characterize the cell death and survival pathways activated in response to mitochondrial dysfunction in non-malignant and cancer cells. The planned studies will provide novel mechanistic insights into the pathways that maintain the mitochondrial genome. This is highly relevant to human health given the evidence linking mutations in the mitochondrial genome with degenerative diseases and ageing. In addition, the proposed studies will provide the framework and rationale for the development of novel therapeutic strategies that selectively target cancer cell mitochondria.